BIOREMEDIATIONBIOREMEDIATIONE. J. del RosarioE. J. del Rosario11, V.P. Migo, V.P. Migo22, M.L.F. , M.L.F. PajePaje

and A.K. Raymundoand A.K. Raymundo33

11Institute of ChemistryInstitute of Chemistry22National Institute of Molecular Biology and National Institute of Molecular Biology and

Biotechnology Biotechnology andand

33Institute of Biological SciencesInstitute of Biological Sciences

University of the Philippines Los Banos, Laguna University of the Philippines Los Banos, Laguna PhilippinesPhilippines

OUTLINE OF PRESENTATIONOUTLINE OF PRESENTATION

1. Introduction1. Introduction1.1. Definitions and concepts1.1. Definitions and concepts1.2. Factors that affect bioremediation1.2. Factors that affect bioremediation

2. Microbiological Aspects2. Microbiological Aspects2.1. Genetics2.1. Genetics2.2. Physiology2.2. Physiology2.3. 2.3. EnzymologyEnzymology/Biochemistry/Biochemistry

3. 3. PhysicoPhysico--Chemical AspectsChemical Aspects3.1. Chemical reactions during bioremediation 3.1. Chemical reactions during bioremediation 3.2. Basic parameters 3.2. Basic parameters –– temperature, pH, temperature, pH, concentrationsconcentrations3.3. Thermodynamic aspects3.3. Thermodynamic aspects3.4. Kinetic aspects3.4. Kinetic aspects

4. Bioengineering Aspects4. Bioengineering Aspects4.1. In situ bioremediation4.1. In situ bioremediation4.3. Ex situ bioremediation 4.3. Ex situ bioremediation

5. Policy Implications5. Policy Implications5.1 Legislation and government policies5.1 Legislation and government policies5.2 Research and development5.2 Research and development5.3 Training and manpower development5.3 Training and manpower development

What is Bioremediation?What is Bioremediation?

•• BioremediationBioremediation is a treatment process is a treatment process that that uses microorganismsuses microorganisms (bacteria, (bacteria, fungi or yeast) fungi or yeast) or or plantsplants

to break down, or to break down, or degradedegrade, hazardous , hazardous substances into less toxic or nontoxic substances into less toxic or nontoxic substances.substances.

PhytoremediationPhytoremediation•• PhytoPhyto means plant; means plant; remediateremediate means means ““to clean to clean

upup””

•• The use of vegetation for The use of vegetation for in situin situ treatment of treatment of contaminated soils, sediments, and water contaminated soils, sediments, and water through contaminant removal, degradation, or through contaminant removal, degradation, or containment.containment.

•• It can be used to clean up metals, pesticides, It can be used to clean up metals, pesticides, solvents, explosives, crude oil, solvents, explosives, crude oil, polyaromaticpolyaromaticHC, and landfill HC, and landfill leachatesleachates..

POLLUTANT

BIOREMEDIATIONPRODUCT(S)

Physico-chemical Factors

•Temperature•pH•Water•O2 / Redox potential•Mass transfer•Solubility

Microbial ParametersGenetic characteristicsPhysiological propertiesMetabolic diversityEnzymologicalcapabilities

Schematic Diagram of Bioremediation Process

Disappearance of 2,4,5-T in soil from the Philippines (PH), Trinidad (TR), Nigeria (NI), and Puerto Rico (PR). (From Rosenberg and Alexander, 1980. Reprinted with permission from the American Chemical Society.)(Martin Alexander)

MICROBIOLOGICAL MICROBIOLOGICAL ASPECTSASPECTS

Examples of Microorganisms Used for Examples of Microorganisms Used for BioremediationBioremediation

•• CaulobacterCaulobacter crescentuscrescentus:: potential for potential for heavyheavy--metal remediation in wastemetal remediation in waste--treatment plant wastewater.treatment plant wastewater.

•• DeinococcusDeinococcus radioduransradiodurans:: Have DNAHave DNA--reparepacapabilities for radioactivecapabilities for radioactivewaste cleanup. waste cleanup.

Ferroplasma Ferroplasma acidarmanusacidarmanus: oxidizes : oxidizes iron, and transforms sulfide in metal iron, and transforms sulfide in metal ores to sulfuric acid.ores to sulfuric acid.

PhytoextractionPhytoextraction

PhytoextractionPhytoextraction employs employs hyperaccumulatinghyperaccumulating plants to remove plants to remove metals from the soil by absorption into the roots and shoots of metals from the soil by absorption into the roots and shoots of the plantthe plant

Genetic engineeringGenetic engineering –– introduction introduction of of gene(sgene(s) for necessary ) for necessary bioremediation bioremediation enzyme(senzyme(s) in ) in genome of host genome of host microrganismmicrorganism..

CHEMICAL ASPECTSCHEMICAL ASPECTS

Chemical Classes and Their Susceptibility to Chemical Classes and Their Susceptibility to BioremediationBioremediationCHEMICAL CLASS EXAMPLES BIODEGRADABILITY

Aromatic Hydrocarbons

Benzene, toluene Aerobic and anaerobic

Ketones and esters Acetone, MEK Aerobic and anaerobic

Petroleum hydrocarbons

Fuel oil Aerobic

Chlorinated solvents TCE, PCE Aerobic (methanotrophs), anaerobic (reductive dechlorination)

Polyaromatichydrocarbons

Anthracene, benzo[a]pyrene, creosote

Aerobic

Naphthalene Phenanthrene

Pyrene Benzo(a)pyrene

Some common polycyclic aromatic hydrocarbons (PAHs)

Initial Degradation of Benzene

COOH

OH

CH2OH

OH

COOHCOOH

OH

catechol 2-hydroxymuconic semialdehyde

2-oxopent-2,4-dienoate(enol form)

4-hydroxy-2-oxovalerate

CH3

COH

acetaldehyde

COOH

O

CH3

C

pyruvate

CH3 COOH

OHO+

Detoxication of Catechol: Aromatic ring cleavage

Microbial Degradation of Microbial Degradation of HCsHCs

C

CCl2

ClCl

H

Cl C

CCl2

Cl

DDT DDECl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

2,3,4,5,6-PENTACHLORO-1-CYCLOHEXENE

LINDANE

CH3CCOOH

Cl

ClCH3CCOOH

O

DALAPON PYRUVIC ACID

Detoxification of Pesticides through Dehalogenation

EXXON VALDEZ (1989)EXXON VALDEZ (1989)

•• Oil spill occurred in ALASKAOil spill occurred in ALASKA•• Tanker loaded with 53 million gallons of oilTanker loaded with 53 million gallons of oil•• Spilled about 35 million gallonsSpilled about 35 million gallons•• Tanker was not doubleTanker was not double--hulled!!!hulled!!!

Oil Slick from

EXXON VALDEZ

GUIMARAS OIL SPILL (2006)GUIMARAS OIL SPILL (2006)

•• M/T Solar I tanker loaded with 2.4 million M/T Solar I tanker loaded with 2.4 million liters of BUNKER OILliters of BUNKER OIL

•• Sunk on August 11, 2006 at Sunk on August 11, 2006 at GuimarasGuimarascoastcoast

•• About 300,000 L oil was spilledAbout 300,000 L oil was spilled•• Worst oil spill in the PhilippinesWorst oil spill in the Philippines

GUIMARAS

GUIMARAS OIL SPILLGUIMARAS OIL SPILL

Affected:Affected:•• 97% of the 220 km97% of the 220 km--long coastlinelong coastline•• 1,143 ha marine reserves1,143 ha marine reserves•• 478 ha mangroves478 ha mangroves•• 16 m2 coral reef16 m2 coral reef

BIOSTIMULATIONBIOSTIMULATION

•• Modification of the contaminated siteModification of the contaminated site•• Addition of fertilizers/nutrientsAddition of fertilizers/nutrients•• Stimulates activity of indigenous HCStimulates activity of indigenous HC

degraders (degraders (IHDsIHDs))•• Balances C:N:P ratioBalances C:N:P ratio•• Not all microbes at site are Not all microbes at site are IHDsIHDs!!!!!!

BIOAUGMENTATIONBIOAUGMENTATION

•• Seeding/introduction of a consortia of Seeding/introduction of a consortia of microorganisms to contaminated sitesmicroorganisms to contaminated sites

•• Microorganisms are laboratoryMicroorganisms are laboratory--testedtestedfor hydrocarbon degradationfor hydrocarbon degradation

Conventional Conventional BioremediationBioremediation

Enzymes released by the microbes Enzymes released by the microbes can only attack one surface of the can only attack one surface of the contaminant. This leads to slower, contaminant. This leads to slower, less effective remediation.less effective remediation.

CleanEARTH'SCleanEARTH'S EnhancedEnhancedBioremediationBioremediationA patented combination of A patented combination of surfactants and emulsifiers break the surfactants and emulsifiers break the contaminant down into tiny pieces, contaminant down into tiny pieces, which can then be surrounded by which can then be surrounded by enzymes and quickly digested.enzymes and quickly digested.

•• ShewanellaShewanella oneidensisoneidensis MR1MR1: may degrade : may degrade organic wastes and reduce or sequester a organic wastes and reduce or sequester a range of toxic metals.range of toxic metals.

Examples of Microorganisms Used for Mine Examples of Microorganisms Used for Mine Waste BioremediationWaste Bioremediation

•• ThiobacillusThiobacillus ferrooxidansferrooxidans : used in mining : used in mining industry to sequester iron & sulfide.industry to sequester iron & sulfide.

•• CyanobacteriaCyanobacteria: produces : produces cyanobacterialcyanobacterialmats which are resistant to metals and mats which are resistant to metals and metalloids, also remove these substances metalloids, also remove these substances from the environment.from the environment.

NAST Biorem Team

In Situ Bioremediation of In Situ Bioremediation of SoilSoil••In situ techniques In situ techniques do not require do not require excavationexcavation of the contaminated of the contaminated soils so may be less expensive, soils so may be less expensive, create less dust, and cause less create less dust, and cause less release of contaminants than ex release of contaminants than ex situ techniques.situ techniques.

Ex Situ Bioremediation of Ex Situ Bioremediation of SoilSoil•• Ex situ techniques Ex situ techniques can be faster, easier to can be faster, easier to

control, and used to treat a wider range of control, and used to treat a wider range of contaminants and soil typescontaminants and soil types than in situ than in situ techniques. However, they techniques. However, they require excavationrequire excavationand treatment of the contaminated soil before and treatment of the contaminated soil before and, sometimes, after the actual and, sometimes, after the actual bioremediation step. Ex situ techniques include bioremediation step. Ex situ techniques include slurryslurry--phase bioremediation phase bioremediation and and solidsolid--phase bioremediation.phase bioremediation.

POLICY IMPLICATIONSPOLICY IMPLICATIONS

PHILIPPINES: TASK FORCE ON BIOREMEDIATION (National Academy of

Science and Technology)To apply scientific and technical expertise to address the problem of toxic and hazardous waste

To design a research program on bioremediation

To develop the capabilities of local scientists and create a pool of expertise to tackle similar problems in the future

R & DR & D: Removing Heavy Metals : Removing Heavy Metals Using Bacterial Using Bacterial

ExopolysaccharidesExopolysaccharides (EPS)(EPS)National Institute of Molecular Biology National Institute of Molecular Biology

and Biotechnology (BIOTECH)and Biotechnology (BIOTECH)University of the Philippines Los BaUniversity of the Philippines Los Baññosos

EPS is produced cheaply from indigenous bacteria (Rhizobium sp.) by fermentation in coconut wastewater medium

EPS removes heavy metals viaadsorption or flocculation andcoagulation processes.

The technology is simple, fastand efficiently removes theheavy metal contaminant

R & DR & D: BIOREMEDIATION : BIOREMEDIATION OF DISTILLERY WASTESOF DISTILLERY WASTES

••Microbial decolorizationMicrobial decolorization••Field application as fertilizerField application as fertilizer

SyntheticSynthetic MelanoidinMelanoidin NaturalNatural MelanoidinMelanoidin

AspergillusAspergillus BacillusBacillus AspergillusAspergillus BacillusBacillus

Absorbance Absorbance (nm)(nm)

InitialInitial 0.5340.534 1.4851.485 0.4000.400 0.5110.511

FinalFinal 0.2170.217 0.4180.418 0.1150.115 0.1990.199

DecolorizationDecolorization%%

59.459.4 71.971.9 71.271.2 61.161.1

Microbial Decolorization of Synthetic and Natural Melanoidins

Desperate Desperate POLLUTIONPOLLUTION calls for calls for desperate desperate BIOREMEDIATION.BIOREMEDIATION.

THANK YOU!THANK YOU!